NEURONAL NICOTINIC ACETYLCHOLINE-RECEPTOR CURRENTS IN PHEOCHROMOCYTOMA (PC12) CELLS - DUAL MECHANISMS OF RECTIFICATION

1. Neuronal nicotinic acetylcholine receptor (nAChR) currents in PC12 cells were studied using single-channel and whole-cell gigaohm seal voltage-clamp techniques. Nicotinic AChR agonists were applied using external pipettes. 2. The single-channel conductance of neuronal nAChRs in outside-out patches under Mg2+-free ionic conditions was 48 pS. In the absence of internal Mg2+ single-channel currents (outside-out patches) did not rectify. 3. Whole-cell nAChR currents recorded with normal internal solution (lacking Mg2+ chelators as described below) were strongly inwardly rectifying. Current vs. voltage relations showed a sharp inflexion near 0 mV, and outward current was never observed. 4. Extensive dialysis with internal solution containing EDTA and Na2-ATP, chelators of intracellular Mg2+ (Mg(i)2+), relieved rectification of instantaneous nAChR currents during voltage jumps from negative potentials. The instantaneous I-V relation became linear with voltage, in agreement with the expectation from single-channel measurements made under similar ionic conditions. 5. Agonist-induced currents recorded under Mg(i)2+-free conditions relaxed towards zero current during depolarizing voltage steps. Rectification of ACh-induced currents at the steady state could be described by a Boltzmann relation, with one-half of channels available at +3.4 mV and an effective gating charge of -0.97. Channel availability was approximately 95% at the resting potential. Opening and closing relaxations under Mg(i)2+-free conditions could be fitted by single exponential functions whose time constants were weakly voltage dependent. 6. A model of rectification was constructed incorporating two voltage-dependent processes: block by Mg(i)2+ and intrinsic channel gating. The I-V relations predicted by this model for both normal and Mg(i)2+-free conditions were in good agreement with the experimental data. We suggest that rectification of nAChR currents in these cells is due to concurrent activity of these two voltage-dependent processes. The relative contributions of these two mechanisms are frequency dependent, with block by Mg(i)2+ dominant for fast events and intrinsic channel gating more important at the steady state.